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Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we use cryo-EM and perform steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III 2 -IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Negatively charged loops of CIII 2 subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allows us to identify water molecules in proton pathways of CIV and, to the best of our knowledge, previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII 2 loops to conduct electron transfer between CIII 2 and CIV., (© 2024. The Author(s).)

Cytochrome c oxidase (CytcO) is an integral membrane protein, which catalyzes four-electron reduction of oxygen linked to proton uptake and pumping. Amphipathic molecules bind in sites near the so-called K proton pathway of CytcO to reversibly modulate its activity. However, purification of CytcO for mechanistic studies typically involves the use of detergents, which may interfere with binding of these regulatory molecules. Here, we investigated the CytcO enzymatic activity as well as intramolecular electron transfer linked to proton transfer upon addition of different detergents to bovine heart mitoplasts. The CytcO activity increased upon addition of alkyl glucosides (DDM and DM) and the steroid analog GDN. The maximum stimulating effect was observed for DDM and DM, and the half-stimulating effect correlated with their CMC values. With GDN the stimulation effect was smaller and occurred at a concentration higher than CMC. A kinetic analysis suggests that the stimulation of activity is due to removal of a ligand bound near the K proton pathway, which indicates that in the native membrane this site is occupied to yield a lower than maximal possible CytcO activity. Possible functional consequences are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aerobic life is powered by membrane-bound redox enzymes that shuttle electrons to oxygen and transfer protons across a biological membrane. Structural studies suggest that these energy-transducing enzymes operate as higher-order supercomplexes, but their functional role remains poorly understood and highly debated. Here we resolve the functional dynamics of the 0.7 MDa III 2 IV 2 obligate supercomplex from Mycobacterium smegmatis, a close relative of M. tuberculosis, the causative agent of tuberculosis. By combining computational, biochemical, and high-resolution (2.3 Å) cryo-electron microscopy experiments, we show how the mycobacterial supercomplex catalyses long-range charge transport from its menaquinol oxidation site to the binuclear active site for oxygen reduction. Our data reveal proton and electron pathways responsible for the charge transfer reactions, mechanistic principles of the quinone catalysis, and how unique molecular adaptations, water molecules, and lipid interactions enable the proton-coupled electron transfer (PCET) reactions. Our combined findings provide a mechanistic blueprint of mycobacterial supercomplexes and a basis for developing drugs against pathogenic bacteria., (© 2024. The Author(s).)

Background: Acne fulminans (AF) is a rare severe acne entity. Although occasionally reported, it is unclear whether AF development is associated with oral isotretinoin treatment., Objectives: To investigate the occurrence of isotretinoin-associated AF, clinical characteristics and prognosis at follow-up., Methods: An international, multicentre, retrospective study was performed in eight hospitals following the call of the EADV Task Force on Acne, Rosacea and Hidradenitis Suppurativa (ARHS). Characteristics of patients treated with isotretinoin before the development of AF (isotretinoin-associated acne fulminans, IAF) were compared with non-IAF (NAF)., Results: Forty-nine patients diagnosed with AF from 2008 to 2022 were included (mean age 16.4 years, SD 2.9, 77.6% male). Αrthralgias/arthritis occurred in 11 patients (22.9%). AF occurred without any previous acne treatment in 26.5% of the patients. Overall, 28 patients (57.1%) developed AF after oral isotretinoin intake (IAF group), while the remaining 21 patients (42.9%) developed AF without previous oral isotretinoin administration (NAF group). IAF occurred after a median duration of isotretinoin treatment of 45 days (IQR: 30, 90). Patients with IAF were more frequently male compared to patients with NAF (89.3% vs. 61.9%, respectively, p = 0.023). There were no differences in patients with IAF versus NAF in patient age, the duration of pre-existing acne, a family history of AF, the distribution of AF lesions or the presence of systemic symptoms or arthralgias. Regarding the management of AF, patients with IAF were treated more frequently with prednisolone (96.2%) compared to those with NAF (70%; p = 0.033) and less frequently with isotretinoin (32.1%) compared to NAF (85.7%; p < 0.001). At a median follow-up of 2.2 years, 76.4% of patients were free of AF and scarring was present in all patients., Conclusions: No specific clinical or demographic characteristics of IAF compared with NAF could be detected, a fact that does not support IAF as a district clinical entity., (© 2023 European Academy of Dermatology and Venereology.)

The respiratory chain in aerobic organisms is composed of a number of membrane-bound protein complexes that link electron transfer to proton translocation across the membrane. In mitochondria, the final electron acceptor, complex IV (CIV), receives electrons from dimeric complex III (CIII 2 ), via a mobile electron carrier, cytochrome c . In the present study, we isolated the CIII 2 CIV supercomplex from the fission yeast Schizosaccharomyces pombe and determined its structure with bound cyt. c using single-particle electron cryomicroscopy. A respiratory supercomplex factor 2 was found to be bound at CIV distally positioned in the supercomplex. In addition to the redox-active metal sites, we found a metal ion, presumably Zn 2+ , coordinated in the CIII subunit Cor1, which is encoded by the same gene ( qcr 1 ) as the mitochondrial-processing peptidase subunit β. Our data show that the isolated CIII 2 CIV supercomplex displays proteolytic activity suggesting a dual role of CIII 2 in S. pombe . As in the supercomplex from S. cerevisiae , subunit Cox5 of CIV faces towards one CIII monomer, but in S. pombe, the two complexes are rotated relative to each other by ~45°. This orientation yields equal distances between the cyt. c binding sites at CIV and at each of the two CIII monomers. The structure shows cyt. c bound at four positions, but only along one of the two symmetrical branches. Overall, this combined structural and functional study reveals the integration of peptidase activity with the CIII 2 respiratory system and indicates a two-dimensional cyt. c diffusion mechanism within the CIII 2 -CIV supercomplex.

Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc 1 ) and one of the organism's terminal oxidases, cytochrome cbb 3 , which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c 4 and a single heme cytochrome c 5 . Together, these proteins allow electron transfer from ubiquinol in cytochrome bc 1 to oxygen in cytochrome cbb 3 . We also present evidence that different isoforms of cytochrome cbb 3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc 1 - cbb 3 SCs also exist in other bacterial pathogens.

Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found in low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the "orphan" two-component response regulator protein MSMEG&#95;2064 as a subunit of the assembly. MSMEG&#95;2064 in the complex occupies a site similar to the proposed redox-sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel, comparable to ubiquinone in other structures and suggesting a conserved quinone binding mechanism.

Fission yeast Schizosaccharomyces pombe serves as model organism for studying higher eukaryotes. We combined the use of cryo-EM and spectroscopy to investigate the structure and function of affinity purified respiratory complex IV (CIV) from S. pombe. The reaction sequence of the reduced enzyme with O 2 proceeds over a time scale of µs-ms, similar to that of the mammalian CIV. The cryo-EM structure of CIV revealed eleven subunits as well as a bound hypoxia-induced gene 1 (Hig1) domain of respiratory supercomplex factor 2 (Rcf2). These results suggest that binding of Rcf2 does not require the presence of a CIII-CIV supercomplex, i.e. Rcf2 is a component of CIV. An AlphaFold-Multimer model suggests that the Hig1 domains of both Rcf1 and Rcf2 bind at the same site of CIV suggesting that their binding is mutually exclusive. Furthermore, the differential functional effect of Rcf1 or Rcf2 is presumably caused by interactions of CIV with their different non-Hig1 domain parts., (© 2023. The Author(s).)

Background: The COVID-19 pandemic has raised questions regarding the management of chronic skin diseases, especially in patients on systemic treatments. Data concerning the use of biologics in adults with psoriasis are reassuring, but data specific to children are missing. Moreover, COVID-19 could impact the course of psoriasis in children., Objectives: The aim of this study was therefore to assess the impact of COVID-19 on the psoriasis of children, and the severity of the infection in relation to systemic treatments., Methods: We set up an international registry of paediatric psoriasis patients. Children were included if they were under 18 years of age, had a history of psoriasis, or developed it within 1 month of COVID-19 and had COVID-19 with or without symptoms., Results: One hundred and twenty episodes of COVID-19 in 117 children (mean age: 12.4 years) were reported. The main clinical form of psoriasis was plaque type (69.4%). Most children were without systemic treatment (54.2%); 33 (28.3%) were on biologic therapies, and 24 (20%) on non-biologic systemic drugs. COVID-19 was confirmed in 106 children (88.3%) and 3 children had two COVID-19 infections each. COVID-19 was symptomatic for 75 children (62.5%) with a mean duration of 6.5 days, significantly longer for children on non-biologic systemic treatments (P = 0.02) and without systemic treatment (P = 0.006) when compared with children on biologics. The six children who required hospitalization were more frequently under non-biologic systemic treatment when compared with the other children (P = 0.01), and particularly under methotrexate (P = 0.03). After COVID-19, the psoriasis worsened in 17 cases (15.2%). Nine children (8%) developed a psoriasis in the month following COVID-19, mainly a guttate form (P = 0.01)., Discussion: Biologics appear to be safe with no increased risk of severe form of COVID-19 in children with psoriasis. COVID-19 was responsible for the development of psoriasis or the worsening of a known psoriasis for some children., (© 2022 European Academy of Dermatology and Venereology.)

(1) Background: Isotretinoin (ISO) is a systemic retinoid known for its teratogenic effects on embryos and fetuses. The aim of this study was to compare the pregnancy outcomes of women who were exposed to isotretinoin with those of women without such exposure from a teratogenic point of view. (2) Methods: A total of 1459 female patients from three clinical hospitals in Poland and Romania, segregated into two groups depending on their ISO exposure, were evaluated between January and December 2019. Medical records were screened to identify the pregnancy outcomes and congenital malformation rates. (3) Results: The congenital malformation rate for the exposed group was 1.2% (four cases), and no specific signs of Accutane embryopathy were identified. Women from the unexposed group were more likely to deliver preterm and through cesarean deliveries and had a higher rate of newborn congenital malformations as compared to women from the exposed group. (4) Conclusions: Even though we could not find a significant association between ISO exposure and teratogenic effects in newborns, effective contraceptive measures are key to preventing unfavorable pregnancy outcomes.

The M. smegmatis respiratory III 2 IV 2 supercomplex consists of a complex III (CIII) dimer flanked on each side by a complex IV (CIV) monomer, electronically connected by a di-heme cyt. cc subunit of CIII. The supercomplex displays a quinol oxidation‑oxygen reduction activity of ~90 e - /s. In the current work we have investigated the kinetics of electron and proton transfer upon reaction of the reduced supercomplex with molecular oxygen. The data show that, as with canonical CIV, oxidation of reduced CIV at pH 7 occurs in three resolved components with time constants ~30 μs, 100 μs and 4 ms, associated with the formation of the so-called peroxy (P), ferryl (F) and oxidized (O) intermediates, respectively. Electron transfer from cyt. cc to the primary electron acceptor of CIV, Cu A , displays a time constant of ≤100 μs, while re-reduction of cyt. cc by heme b occurs with a time constant of ~4 ms. In contrast to canonical CIV, neither the P → F nor the F → O reactions are pH dependent, but the P → F reaction displays a H/D kinetic isotope effect of ~3. Proton uptake through the D pathway in CIV displays a single time constant of ~4 ms, i.e. a factor of ~40 slower than with canonical CIV. The slowed proton uptake kinetics and absence of pH dependence are attributed to binding of a loop from the QcrB subunit of CIII at the D proton pathway of CIV. Hence, the data suggest that function of CIV is modulated by way of supramolecular interactions with CIII., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

SARS-CoV-2 is the cause of COVID-19 disease and responsible for a pandemic since the 2020. Multiple organ involvement has been described including cutaneous symptoms. Affection of skin appendages, however, seems to be under-reported except for COVID-toes. We performed a PUBMED research for "COVID-19" OR "SARS-CoV-2" AND "skin appendages", "hair", "nails", and "skin glands" from January 2020 to April 2022. COVID toes were excluded since this symptom had extensively been discussed. The focus of this narrative review was laid on clinical presentation, association to the course of COVID-19 disease and treatment options. Skin appendages can be affected by COVID-19 disease beyond COVID-toes, both by symptomatic and asymptomatic course. Telogen effluvium, androgenetic alopecia, and alopecia areata are the most common hair disorders in COVID-19 patients. Nails are less commonly affected by COVID-19 than hair. Splinter hemorrhages and leukonychia are the most frequent findings. While sebaceous glands seem to be uninvolved, SARS-CoV-2 spike proteins have been identified in eccrine sweat glands. Alopecia areata is often seen among asymptomatic COVID-19 patients while telogen effluvium is observed in symptomatic and asymptomatic patients. The half-moon sign on the nails could be a red flag for a more severe course of COVID-19. Treatment options are summarized. Skin appendages are not spared by COVID-19. Their knowledge will help to identify asymptomatic patients and patients at risk for a more severe course of the viral disease.

Introduction: Acne vulgaris is a chronic inflammatory skin disease of the pilosebaceous follicles that affects patients of all ages., Aim: Use of isotretinoin in the early stages of the disease to prevent subsequent lesions of acne, including prolonged treatment and acne scars at a later age., Material and Methods: A retrospective, comparative study was carried between January 2010 and November 2018. The study population consisted of 90 children aged 9-18 years with acne. During treatment by isotretinoin the clinical evaluation was done every month. Patients were divided into three groups according to age. One of the qualification criteria was follow-up visits., Results: A total of 90 children (67.8% females; mean age: 13.5 years) were enrolled. In group A (30 individuals - aged 9-11) and B (30 individuals - aged 12-13), treatment was terminated 2 months after clinical improvement (mean: 3 months). In control group C (30 individuals - aged 14-18), treatment was carried out using average cumulative dose 135 mg/kg bw/day. All groups showed up for follow-up. after 1 to 8 years. In groups A and B, 13 people underwent a second acne treatment; in 3.33% oral isotretinoin was used, in 18.33% topical treatment. In group C, 30 (100%) individuals underwent a second acne treatment; in 20% oral isotretinoin was used, and 80% required a topical treatment. Acne scars and post acne hyperpigmentation have been documented in 73.33% in group C., Conclusions: Early, reasonable and short-term use of isotretinoin can reduce the incidence of acne in the future and reduce the occurrence of secondary acne symptoms., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2022 Termedia Sp. z o. o.)

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme's catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

The apple maggot, Rhagoletis pomonella (Walsh), was introduced into the apple-growing regions of the Pacific Northwest in the U.S.A. during the past 60-100 yr. Apple maggot (larvae, puparia, and adults) is difficult to distinguish from its morphologically similar sister species, Rhagoletis zephyria Snow, which is native and abundant in the Pacific Northwest. While morphological identifications are common practice, a simple, inexpensive assay based on genetic differences would be very useful when morphological traits are unclear. Here we report nucleotide substitution and insertion-deletion mutations in the nontranscribed spacer (NTS) of the ribosomal RNA gene cistron of R. pomonella and R. zephyria that appear to be diagnostic for these two fly species. Insertion-deletion variation is substantial and results in a 49 base-pair difference in PCR amplicon size between R. zephyria and R. pomonella that can be scored using agarose gel electrophoresis. PCR amplification and DNA sequencing of 766 bp of the NTS region from 38 R. pomonella individuals and 35 R. zephyria individuals from across their geographic ranges led to the expected PCR fragments of approx. 840 bp and 790 bp, respectively, as did amplification and sequencing of a smaller set of 26 R. pomonella and 16 R. zephyria flies from a sympatric site in Washington State. Conversely, 633 bp mitochondrial COI barcode sequences from this set of flies were polyphyletic with respect to R. pomonella and R. zephyria. Thus, differences in NTS PCR products on agarose gels potentially provide a simple way to distinguish between R. pomonella and R. zephyria., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII 2 CIV 2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII 2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the Q N and Q P sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme b L . A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme b L . Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vaccination against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV‑2) has become a major tool in the battle against the coronavirus disease 2019 (COVID-19) pandemic. Numerous products have been developed and more are to come. Vaccination success varies greatly between different countries. There are a number of different vaccine types, such as mRNA, DNA vaccines, adenovirus vector vaccines, and full-length spike protein nanoparticles with a special matrix. The different types may also cause a different spectrum of adverse events. With mass vaccination, post-marketing surveillance for product safety becomes increasingly important. In this review, we discuss possible hypersensitivity and cutaneous adverse events related to SARS-CoV‑2 vaccination-from local reactions like COVID arm to systemic and severe reactions like anaphylaxis. Vaccination may also induce or exacerbate preexisting disorders such as herpes zoster infection. This review should provide information to tailor, whenever possible, vaccination to patients' needs. It is a contribution to patient safety as well. There is general consensus that the benefits of SARS-CoV‑2 vaccination currently outweigh the risks of possible adverse events., (© 2021. Springer-Verlag GmbH Austria, ein Teil von Springer Nature.)

Electron transfer between respiratory complexes drives transmembrane proton translocation, which powers ATP synthesis and membrane transport. The homodimeric respiratory complex III (CIII 2 ) oxidizes ubiquinol to ubiquinone, transferring electrons to cytochrome c and translocating protons through a mechanism known as the Q cycle. The Q cycle involves ubiquinol oxidation and ubiquinone reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. We determined structures of Candida albicans CIII 2 by cryoelectron microscopy (cryo-EM), revealing endogenous ubiquinone and visualizing the continuum of Rieske head domain conformations. Analysis of these conformations does not indicate cooperativity in the Rieske head domain position or ligand binding in the two CIIIs of the CIII 2 dimer. Cryo-EM with the indazole derivative Inz-5, which inhibits fungal CIII 2 and is fungicidal when administered with fungistatic azole drugs, showed that Inz-5 inhibition alters the equilibrium of Rieske head domain positions., Competing Interests: Declaration of interests J.L.R. is an adviser and shareholder for Structura Biotechnology, which develops the cryoSPARC software package used here. L.E.C. and L.W. are co-founders of and shareholders in Bright Angel Therapeutics, a platform company for development of antifungal therapeutics. L.E.C. is a consultant for Boragen, a small-molecule development company focused on boron chemistry for crop protection and animal health. L.W. is an inventor on US patent 9738610 describing the synthesis and use of indazoles as antimicrobials., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than 50 years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII 2 CIV 2 ). In mycobacterial electron transport chains, CIII 2 CIV 2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII 2 CIV 2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII 2 CIV 2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII 2 CIV 2 activity., Competing Interests: DY, JD, SK, RA, SB, PI, PB, JR No competing interests declared, (© 2021, Yanofsky et al.)

In the final steps of energy conservation in aerobic organisms, free energy from electron transfer through the respiratory chain is transduced into a proton electrochemical gradient across a membrane. In mitochondria and many bacteria, reduction of the dioxygen electron acceptor is catalyzed by cytochrome c oxidase (complex IV), which receives electrons from cytochrome bc 1 (complex III), via membrane-bound or water-soluble cytochrome c . These complexes function independently, but in many organisms they associate to form supercomplexes. Here, we review the structural features and the functional significance of the nonobligate III 2 IV 1/2 Saccharomyces cerevisiae mitochondrial supercomplex as well as the obligate III 2 IV 2 supercomplex from actinobacteria. The analysis is centered around the Q-cycle of complex III, proton uptake by Cyt c O, as well as mechanistic and structural solutions to the electronic link between complexes III and IV.

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc 1 and aa 3 -type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O 2 reduction activity of the cyt. bc 1 -aa 3 supercomplex. The potential role of the membrane-anchored cyt. c y as a component in supercomplexes was also investigated., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Mitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker's yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex., Results: Here, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility., Conclusions: Together with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

Energy conversion in aerobic organisms involves an electron current from low-potential donors, such as NADH and succinate, to dioxygen through the membrane-bound respiratory chain. Electron transfer is coupled to transmembrane proton transport, which maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred from respiratory complexes III to IV (CIII and CIV) by water-soluble cytochrome (cyt.) c In Saccharomyces cerevisiae and some other organisms, these complexes assemble into larger CIII 2 CIV 1/2 supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the S. cerevisiae supercomplex cyt. c -mediated QH 2 :O 2 oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Single-particle electron cryomicroscopy (cryo-EM) structures of the supercomplex with cyt. c show the positively charged cyt. c bound to either CIII or CIV or along a continuum of intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively charged patch on the supercomplex surface. Thus, rather than enhancing electron transfer rates by decreasing the distance that cyt. c must diffuse in three dimensions, formation of the CIII 2 CIV 1/2 supercomplex facilitates electron transfer by two-dimensional (2D) diffusion of cyt. c This mechanism enables the CIII 2 CIV 1/2 supercomplex to increase QH 2 :O 2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain., Competing Interests: The authors declare no competing interest.

The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc 1 and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble α helices and five putative transmembrane (TM) α helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation. The NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hidradenitis suppurativa/acne inversa (HS/AI) is one of the most debilitating dermatoses with a strong negative impact on every dimension of quality of life. Treatment is dependent on the severity of clinical manifestations and comorbidities. While anti-inflammatory and antimicrobial approaches are recommended for mild and moderate stages, immunomodulatory drugs have gained increasing interest in all stages of HS/AI. We reviewed the available data on this subject in a narrative review and included not only substances with published final outcome but those where either the ongoing trials or experience from case report. Furthermore, we investigated combined surgical therapy and immunomodulatory drugs and raised specific questions to be answered in controlled settings. This aspect seems to be underrepresented. The first approved medical treatment for HS/AI is adalimumab. Other cytokine, interleukin, Janus kinase and C5a inhibitors and antagonists are under investigation. IL-1 inhibitors and antagonists may become an option for mild to moderate HS/AI, while most of the other medical compounds target moderate to severe HS/AI. Despite medical efforts with immunomodulatory agents, surgery remains a cornerstone of efficient HS/AI therapy. Better outcome in advanced disease might be achieved by combining drug therapy and surgery, but more systematic clinical trials are necessary for the optimal combination., (© 2020 Wiley Periodicals LLC.)

The transmembrane protein cytochrome c oxidase (C c O) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. C c O is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in C c O, while studies reporting proton turnover rates of C c O, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute C c O from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. C c O proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted C c Os across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli , as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the C c O proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active C c Os. In particular, our experiments indicate that efficient functional reconstitution of C c O is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

Components of respiratory chains in mitochondria and some aerobic bacteria assemble into larger, multiprotein membrane-bound supercomplexes. Here, we address the functional significance of supercomplexes composed of respiratory-chain complexes III and IV. Complex III catalyzes oxidation of quinol and reduction of water-soluble cytochrome c (cyt c), while complex IV catalyzes oxidation of the reduced cyt c and reduction of dioxygen to water. We focus on two questions: (i) under which conditions does diffusion of cyt c become rate limiting for electron transfer between these two complexes? (ii) is there a kinetic advantage of forming a supercomplex composed of complexes III and IV? To answer these questions, we use a theoretical approach and assume that cyt c diffuses in the water phase while complexes III and IV either diffuse independently in the two dimensions of the membrane or form supercomplexes. The analysis shows that the electron flux between complexes III and IV is determined by the equilibration time of cyt c within the volume of the intermembrane space, rather than the cyt c diffusion time constant. Assuming realistic relative concentrations of membrane-bound components and cyt c and that all components diffuse independently, the data indicate that electron transfer between complexes III and IV can become rate limiting. Hence, there is a kinetic advantage of bringing complexes III and IV together in the membrane to form supercomplexes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Cytochrome c oxidase is a membrane-bound redox-driven proton pump that harbors two proton-transfer pathways, D and K, which are used at different stages of the reaction cycle. Here, we address the question if a D pathway with a modified energy landscape for proton transfer could take over the role of the K pathway when the latter is blocked by a mutation. Our data indicate that structural alterations near the entrance of the D pathway modulate energy barriers that influence proton transfer to the proton-loading site. The data also suggest that during reduction of the catalytic site, its protonation has to occur via the K pathway and that this proton transfer to the catalytic site cannot take place through the D pathway., (© 2019 Federation of European Biochemical Societies.)

Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O 2 -reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Å from the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mitochondrial respiratory chain is assembled into supercomplexes. Previously, two respiratory supercomplex-associated proteins, Rcf1 and Rcf2, were identified in Saccharomyces cerevisiae , which were initially suggested to mediate supercomplex formation. Recent evidence suggests that these factors instead are involved in cytochrome c oxidase biogenesis. We demonstrate here that Rcf1 mediates proper function of cytochrome c oxidase, while binding of Rcf2 results in a decrease of cytochrome c oxidase activity. Chemical crosslink experiments demonstrate that the conserved Hig-domain as well as the fungi specific C-terminus of Rcf1 are involved in molecular interactions with the cytochrome c oxidase subunit Cox3. We propose that Rcf1 modulates cytochrome c oxidase activity by direct binding to the oxidase to trigger changes in subunit Cox1, which harbors the catalytic site. Additionally, Rcf1 interaction with cytochrome c oxidase in the supercomplexes increases under respiratory conditions. These observations indicate that Rcf1 could enable the tuning of the respiratory chain depending on metabolic needs or repair damages at the catalytic site., (Copyright © 2020 Dawitz, Schäfer, Schaart, Magits, Brzezinski and Ott.)

Mitochondrial energy conversion involves a chain of membrane-bound proteins that are wired to conduct an electron current, which drives transmembrane proton translocation. These enzymes associate to form supercomplexes, but the functional relevance of the higher-order structures is unknown. A recent study by Letts et al. presents structures of a supercomplex, which suggest how the interaction choreography may control overall functionality., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In cytochrome c oxidase (CytcO) reduction of O 2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than through the D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D (Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO with O 2 . No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and presumably also for proton pumping.

Cutaneous melanoma is an aggressive neoplasia of melanocytes. Prognosis is dependent on tumor stage. Stage IV melanoma is characterized by the occurrence of distant metastases. Response of metastases to classical chemotherapy is limited and toxicity of treatment is high. In recent years, new developments in immunotherapy and targeted therapies improved prognosis of stage IV melanoma patients with better tolerability of treatment. There is no dispute about surgical treatment of primary melanoma. But what is the value of metastasectomy in the era of new systemic treatments? This review aims to discuss available data for surgical removal of distant metastases for several organs and tissues. The available evidence suggests that for selected patients with possible complete resection of all tumor metastases, metastasectomy remains an effective treatment option with a benefit in overall survival.

Aesthetic dermatology (AD) is a rapidly growing subspecialty of dermatology. The acceptance of AD in scientific community and the society is associated with its competence, efficiency, and seriousness. This review highlights some recent developments toward new tools, techniques, and understanding in the field of AD. Analyzing the specific needs of patients and assessing the effect by objective measurements is important for further progress. For long time ignored, white adipose tissue has gained increasing interest in biology and rejuvenation. Characterization of dermal and subcutaneous white adipose tissue has made progress. The interaction of hyaluronic acid and calcium hydroxyl apatite (CaHA) fillers with adipocytes could be responsible for clinical efficacy. New developments of oral collagen treatment and highly diluted CaHA to contour the body outside the face will be discussed. Submental contouring using purified desoxycholic acid is another new development., (© 2018 Wiley Periodicals, Inc.)

The cytochrome bo 3 quinol oxidase from Vitreoscilla (vbo 3 ) catalyses oxidation of ubiquinol and reduction of O 2 to H 2 O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo 3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na + on the oxidase activity of purified vbo 3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo 3 does not pump Na + ions, but H + across the membrane, and that H + -pumping is not influenced by the presence of Na + . (iii) Using an oxygen pulse method, it was found that 2 H + /e - are ejected from proteoliposomes, in agreement with the values found for the H + -pumping bo 3 oxidase of Escherichia coli (ecbo 3 ). This coincides with the interpretation that 1 H + /e - is pumped across the membrane and 1 H + /e - is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo 3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na + . Overall the data show that vbo 3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo 3 quinol oxidase.

Respiratory chain complexes execute energy conversion by connecting electron transport with proton translocation over the inner mitochondrial membrane to fuel ATP synthesis. Notably, these complexes form multi-enzyme assemblies known as respiratory supercomplexes. Here we used single-particle cryo-EM to determine the structures of the yeast mitochondrial respiratory supercomplexes III 2 IV and III 2 IV 2 , at 3.2-Å and 3.5-Å resolutions, respectively. We revealed the overall architecture of the supercomplex, which deviates from the previously determined assemblies in mammals; obtained a near-atomic structure of the yeast complex IV; and identified the protein-protein and protein-lipid interactions implicated in supercomplex formation. Take together, our results demonstrate convergent evolution of supercomplexes in mitochondria that, while building similar assemblies, results in substantially different arrangements and structural solutions to support energy conversion.

Competing Interests: There are no conflicts of interest.

In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III 2 IV 2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O 2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Q o and Q i sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c 1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

Functional studies of membrane-bound channels, transporters or signal transducers require that the protein of interest resides in a membrane that separates two compartments. One approach that is commonly used to prepare these systems is to reconstitute the protein in liposomes. An intermediate step of this method is purification of the protein, which typically involves solubilization of the native membrane using detergent. The use of detergents often results in removal of lipids surrounding the protein, which may alter its structure and function. Here, we have employed a method for isolation of membrane proteins with a disc of their native lipids to develop an approach that allows transfer of the purified membrane protein to liposomes without the use of any detergents.

Respiration in Saccharomyces cerevisiae is regulated by small proteins such as the respiratory supercomplex factors (Rcf). One of these factors (Rcf1) has been shown to interact with complexes III (cyt. bc 1 ) and IV (cytochrome c oxidase, CytcO) of the respiratory chain and to modulate the activity of the latter. Here, we investigated the effect of deleting Rcf1 on the functionality of CytcO, purified using a protein C-tag on core subunit 1 (Cox1). Specifically, we measured the kinetics of ligand binding to the CytcO catalytic site, the O 2 -reduction activity and changes in light absorption spectra. We found that upon removal of Rcf1 a fraction of the CytcO is incorrectly assembled with structural changes at the catalytic site. The data indicate that Rcf1 modulates the assembly and activity of CytcO by shifting the equilibrium of structural sub-states toward the fully active, intact form., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.

Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

The respiratory supercomplex factor 1 (Rcf 1) in Saccharomyces cerevisiae binds to intact cytochrome c oxidase (CytcO) and has also been suggested to be an assembly factor of the enzyme. Here, we isolated CytcO from rcf1Δ mitochondria using affinity chromatography and investigated reduction, inter-heme electron transfer and ligand binding to heme a 3 . The data show that removal of Rcf1 yields two CytcO sub-populations. One of these sub-populations exhibits the same functional behavior as CytcO isolated from the wild-type strain, which indicates that intact CytcO is assembled also without Rcf1. In the other sub-population, which was shown previously to display decreased activity and accelerated ligand-binding kinetics, the midpoint potential of the catalytic site was lowered. The lower midpoint potential allowed us to selectively reduce one of the two sub-populations of the rcf1Δ CytcO, which made it possible to investigate the functional behavior of the two CytcO forms separately. We speculate that these functional alterations reflect a mechanism that regulates O 2 binding and trapping in CytcO, thereby altering energy conservation by the enzyme.

Cellular proteostasis is maintained via the coordinated synthesis, maintenance, and breakdown of proteins in the cytosol and organelles. While biogenesis of the mitochondrial membrane complexes that execute oxidative phosphorylation depends on cytoplasmic translation, it is unknown how translation within mitochondria impacts cytoplasmic proteostasis and nuclear gene expression. Here we have analyzed the effects of mutations in the highly conserved accuracy center of the yeast mitoribosome. Decreased accuracy of mitochondrial translation shortened chronological lifespan, impaired management of cytosolic protein aggregates, and elicited a general transcriptional stress response. In striking contrast, increased accuracy extended lifespan, improved cytosolic aggregate clearance, and suppressed a normally stress-induced, Msn2/4-dependent interorganellar proteostasis transcription program (IPTP) that regulates genes important for mitochondrial proteostasis. Collectively, the data demonstrate that cytosolic protein homeostasis and nuclear stress signaling are controlled by mitochondrial translation efficiency in an inter-connected organelle quality control network that determines cellular lifespan., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cytochrome c oxidase catalyzes reduction of O 2 to H 2 O at a catalytic site that is composed of a copper ion and heme group. The reaction is linked to translocation of four protons across the membrane for each O 2 reduced to water. The free energy associated with electron transfer to the catalytic site is unequal for the four electron-transfer events. Most notably, the free energy associated with reduction of the catalytic site in the oxidized cytochrome c oxidase (state O) is not sufficient for proton pumping across the energized membrane. Yet, this electron transfer is mechanistically linked to proton pumping. To resolve this apparent discrepancy, a high-energy oxidized state (denoted O H ) was postulated and suggested to be populated only during catalytic turnover. The difference between states O and O H was suggested to be manifested in an elevated midpoint potential of Cu B in the latter. This proposal predicts that one-electron reduction of cytochrome c oxidase after its oxidation would yield re-reduction of essentially only Cu B . Here, we investigated this process and found ~5% and ~6% reduction of heme a 3 and Cu B , respectively, i.e. the apparent redox potentials for heme a 3 and Cu B are lower than that of heme a.

The Saccharomyces cerevisiae respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a "charge zipper" motif. The dimer structure is supported by molecular dynamics (MD) simulations in DPC, although the simulations show a more dynamic dimer interface than the NMR data. Furthermore, CD and NMR data indicate that Rcf1 undergoes a structural change when reconstituted in liposomes, which is supported by MD data, suggesting that the dimer structure is unstable in a planar membrane environment. Collectively, these data indicate a dynamic monomer-dimer equilibrium. Furthermore, the Rcf1 dimer interacts with cytochrome c , suggesting a role as an electron-transfer bridge between complexes III and IV. The Rcf1 structure will help in understanding its functional roles at a molecular level., Competing Interests: The authors declare no conflict of interest.

The Saccharomyces cerevisiae mitochondrial respiratory supercomplex factor 2 (Rcf2) plays a role in assembly of supercomplexes composed of cytochrome bc 1 (complex III) and cytochrome c oxidase (complex IV). We expressed the Rcf2 protein in Escherichia coli, refolded it, and reconstituted it into dodecylphosphocholine (DPC) micelles. The structural properties of Rcf2 were studied by solution NMR, and near complete backbone assignment of Rcf2 was achieved. The secondary structure of Rcf2 contains seven helices, of which five are putative transmembrane (TM) helices, including, unexpectedly, a region formed by a charged 20-residue helix at the C terminus. Further studies demonstrated that Rcf2 forms a dimer, and the charged TM helix is involved in this dimer formation. Our results provide a basis for understanding the role of this assembly/regulatory factor in supercomplex formation and function., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)